Live whole Plasmodium falciparum sporozoite (PfSPZ) vaccines are being assessed in clinical trials globally. Sanaria?s lead PfSPZ product, PfSPZ Vaccine, is attenuated by radiation and has been safe and well tolerated in >1,570 infants, children and adults. It has induced >90% vaccine efficacy (VE) against homologous controlled human malaria infection (CHMI) at 3-11 weeks after immunization in the US, Germany, Mali and Tanzania; 80% and 54% VE against heterologous CHMI at 2.5 and 8 months; and VE durable for at least 6 months against intense naturally transmitted Pf malaria in 3 field trials in Africa. Based on this success, PfSPZ Vaccine is advancing to Phase 3 clinical trials and licensure. Because the parasites in PfSPZ Vaccine are non- replicating, it requires a relatively high dose of PfSPZ. Sanaria?s 2nd generation vaccine is PfSPZ-CVac (Chemoprophylaxis Vaccine). PfSPZ-CVac provided 100% VE against CHMI 10 weeks after vaccination using <10% the dose required for PfSPZ Vaccine, and gave 100% VE against heterologous CHMI at 12 weeks despite a 4.5-fold reduction in dose. PfSPZ-CVac improves VE beyond what PfSPZ Vaccine has achieved and concurrently lessens the cost of goods due to the low dose required. This is because the PfSPZ replicate up to 50,000 times in the liver and are killed in the bloodstream by a chemoprophylaxis drug, usually chloroquine. The drawback is the transient parasitemia and associated malaria symptoms that occur 7-8 days after 1st immunization before the parasites are killed. This application supports development of a PfSPZ-based vaccine using genetically attenuated (GA) parasites that, unlike PfSPZ Vaccine, replicate in the liver, greatly amplifying the immune stimulus, and unlike PfSPZ-CVac, arrest late in liver stage development due to the deletion of an essential protein. This late-arresting, GA parasite, Pf?Plasmei2, lacks Plasmei2, the gene encoding the meiosis inhibited 2 RNA binding protein. Mei2 RBP is exclusively transcribed in the liver stage and conserved among Plasmodium species. In the P. yoelii murine malaria model, deletion of Plasmei2 gene caused late liver stage arrest of 95% of the parasites. In the PfSPZ FRG mouse/ humanized liver model, attenuation was 100%. In this approach, Pf?Plasmei2 will be substituted for wild type, fully infectious PfSPZ in PfSPZ-CVac, eliminating transient parasitemia thereby improving both tolerability and safety. If there is any evidence of breakthrough parasites in the first clinical trials, chloroquine will also be given to serve as a safety net to kill any parasites leaking into the blood. Phase I SBIR funds from this grant will be used to characterize the development of this gene deleted strain in humanized FRG huHep mice, optimize methods to produce PfSPZ of Pf?Plasmei2, and manufacture a master cell bank. A pilot lot of Pf?Plasmei2 PfSPZ (Sanaria PfSPZ- LARC1) will be produced to show manufacturability and stability of cryopreserved material. This work will be followed in Phase II by IND-directed activities, production of a GMP clinical lot, IND submission, and under separate funding, conduct of a clinical trial to assess safety, immunogenicity and VE of PfSPZ-LARC1.